Kits, compositions and methods for detecting a biological condition

ABSTRACT

The present invention provides kits, apparatus and methods for determining a biological condition in a mammalian subject, the method includes incubating a specimen from a patient with at least one composition in a kit for a predetermined period of time to form at least one reaction product, when the subject has said biological condition, and receiving an indication of the at least one reaction product responsive to at least one reporter element in the kit thereby providing the indication of the biological condition in the subject.

CROSS-REFERENCE

This application is a continuation application of application Ser. No.14/942,921, filed on Nov. 16, 2015, which is a continuation applicationof Ser. No. 14/571,906, filed on Dec. 16, 2014 and now issued as U.S.Pat. No. 9,207,239, which is a continuation application of applicationSer. No. 14/296,317, filed on Jun. 4, 2014 and now issued as U.S. Pat.No. 8,945,913, which is a divisional application of Ser. No. 13/716,246,filed Dec. 17, 2012, each of which is entirely incorporated herein byreference.

The disclosures of the co-pending US Provisional Patent Application toKasdan, et al, filed on Nov. 17, 2012, and titled “Kits, Compositionsand Methods for Detecting a Biological Condition” and the co-pending USProvisional Patent Application to Kasdan, et al, filed on Nov. 17, 2012,and titled “Kits, Compositions and Methods for Rapid Chemical Detection”are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to apparatus and methods fordetecting a biological condition, and more specifically to methods andapparatus for detecting a biological condition in small fluid samples.

BACKGROUND OF THE INVENTION

There are numerous medical conditions which are hard to diagnose. Oftendiagnosis by a physician is based on the physician's observation ofcombinations of symptoms in a patient. This sometimes leads tomisdiagnosis. Furthermore, the patient's response to a treatment,whether drug or other modality is often followed up by physician'sobservation.

Many laboratory tests are performed in the diagnostic arena on a bodilyspecimen or fluid to determine a biological condition in a patient.However, these tests are performed off-line in diagnostic laboratories.Often, the laboratory services are only provided during a single 8-hourshift during the day and tend to be labor intensive. Some prior artpublications in the field include, inter alia, U.S. Pat. No. 8,116,984,US2006215155 and US2012187117.

Despite the inventions mentioned hereinabove, there still remains anunmet need to provide improved apparatus and methods for detecting anddiagnosing biological conditions in a patient.

SUMMARY OF THE INVENTION

It is an object of some aspects of the present invention to provideimproved apparatus and methods for detecting and diagnosing biologicalconditions in a patient.

In some embodiments of the present invention, improved methods,apparatus and kits are provided for detecting and diagnosing abiological condition in a patient.

In other embodiments of the present invention, a method and kit isdescribed for providing rapid detection of biological moieties in asample from a patient.

In further embodiments of the present invention, a method and kit isdisclosed for providing detection of biological moieties in a smallfluid sample from a patient.

There is thus provided according to an embodiment of the presentinvention, a kit for evaluating a biological condition in a patient, thekit comprising;

-   -   a) a disposable element for receiving a biological specimen and        for combining said specimen with at least one composition;    -   b) at least one composition comprising at least one detector        moiety adapted to react with said specimen to form a reaction        product, when said patient has said biological condition; and    -   c) at least one reporter element adapted to provide an        indication of reaction product thereby providing the indication        of the biological condition. Additionally, according to an        embodiment of the present invention, the kit further comprises;    -   d) instructions for using the kit.

Furthermore, according to an embodiment of the present invention, thedisposable element is a disposable cartridge.

Moreover, according to an embodiment of the present invention, thedisposable cartridge is a disposable microfluidics cartridge.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least one of thefollowing elements:

a) a reservoir;

b) a pump;

c) a valve;

d) a conduit;

e) a motor;

f) a miniaturized flow cell;

g) a transport channel;

h) a microfluidic element;

i) a compressed gas holding element;

j) a compressed gas releasing element;

k) a nozzle element;

l) a mixing element;

m) a bellows element;

n) software adapted to activate said elements according to a specificsequence; and

o) hardware to activate said elements according to a specific sequence.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least two of theelements.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least three of theelements.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least four of theelements.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least five of theelements.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least ten of theelements.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least twenty of theelements.

Additionally, according to an embodiment of the present invention, thedisposable microfluidics cartridge comprises at least thirty of theelements.

According to an embodiment of the present invention, the microfluidicskit is configured to provide the rapid indication with one hour.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication withthirty minutes.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication withfifteen minutes.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication with tenminutes.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication withfive minutes.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication with oneminute.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication withthirty seconds.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication with tenseconds.

According to another embodiment of the present invention, themicrofluidics kit is configured to provide the rapid indication with onesecond.

There is thus provided according to an embodiment of the presentinvention, a microfluidics assay kit for performing a rapid biologicalassay, the kit comprising;

-   -   a) a disposable element comprising a reactant, the disposable        element being adapted to receive a sample comprising a        biological entity and for combining said reactant with said        biological entity to form a reaction product; and    -   b) at least one reporter element adapted to provide a rapid        indication of disappearance of said reactant thereby providing        rapid assay of the biological entity.

There is thus provided according to an embodiment of the presentinvention, a microfluidics assay kit for performing a rapid assay of abiological entity, the kit comprising;

-   -   a) a disposable element comprising a reactant, the disposable        element being adapted to receive a sample comprising the        biological entity and for combining said reactant with said        biological entity to form a reaction product; and    -   b) at least one reporter element adapted to provide a rapid        indication of appearance of said reaction product thereby        providing rapid assay of the biological entity. There is thus        provided according to an embodiment of the present invention, a        composition for evaluating a biological condition, the        composition comprising;        -   a. a sample composition comprising at least one of;            -   i. a bodily specimen comprising a target moiety;            -   ii. a positive control moiety; and            -   iii. a negative control moiety;        -   b. a detection composition comprising at least one of;            -   i. at least one target antibody;            -   ii. at least one positive control identifying antibody;                and            -   iii. at least one negative control identifying detection                moiety or characteristic; and        -   c. at least one reference composition comprising at least            one of;            -   i. a target signal reference composition; and            -   ii. a reference identifier composition.

There is thus provided according to another embodiment of the presentinvention a composition for evaluating a biological condition, thecomposition comprising;

-   -   a. a sample composition comprising at least one of;        -   i. a bodily specimen comprising a target moiety;        -   ii. a positive control moiety; and        -   iii. a negative control moiety;    -   b. an antibody composition comprising at least one of;        -   i. at least one target antibody (CD64 antibody);        -   ii. at least one positive control identifying antibody            (CD163); and        -   iii. at least one negative control identifying antibody or            characteristic; and    -   c. at least one reference composition comprising at least one        of;        -   i. a target signal reference composition; and        -   ii. a reference identifier composition.

Additionally, according to an embodiment of the present invention, thecomposition further comprises at least one conditioning moietycomprising;

-   -   a. at least one lysis reagent; and    -   b. at least one diluent.

Furthermore, according to an embodiment of the present invention, thebiological condition is selected from a group consisting of blooddiseases such as leukemia, thrombocytopenia immune system disorders,local infections, urinary tract disorders, autoimmune diseases andsepsis.

Moreover, according to an embodiment of the present invention the bodilyspecimen is selected from a group consisting of blood, serum, plasma,urine, saliva, cerebrospinal fluid (CSF), serous fluid, peritoneal fluidand synovial fluid.

According to another embodiment of the present invention, the targetmoiety includes a CD64 surface antigen on neutrophils.

Additionally, according to a further embodiment of the presentinvention, the positive control moiety includes monocytes and thenegative control includes lymphocytes. Additionally, according to anembodiment of the present invention, the target moiety is CD64 onneutrophils, the positive control moiety includes CD64 expression onmonocytes, and the negative control moiety includes lymphocytes withoutCD64 expression.

Further, according to an embodiment of the present invention, the targetindicator is bound to a signaling moiety on the at least one targetantibody.

Yet further, according to an embodiment of the present invention, the atleast one reference composition includes beads.

Additionally, according to an embodiment of the present invention, thebeads include polystyrene microbeads.

Moreover, according to an embodiment of the present invention, thetarget antibody reference composition includes a first fluorescentsignal and the reference identifier composition includes a secondfluorescent signal.

Furthermore, according to an embodiment of the present invention, thefirst fluorescent signal includes FITC and the second fluorescent signalincludes Starfire Red fluor.

There is thus provided according to an embodiment of the presentinvention, a method of quantifying a biomarker in a sample, comprising;

-   -   a. contacting the sample with a fluorescently-labeled binding        moiety that specifically binds to the biomarker;    -   b. detecting a first fluorescent signal from at least a portion        of the labeled sample;    -   c. detecting a second fluorescent signal from a population of        fluorescently-labeled particles, wherein the population includes        a known fluorescent intensity over a fixed time; and    -   d. normalizing the first fluorescent signal to the second        fluorescent signal, thereby quantifying the biomarker, wherein        the normalizing includes using a device comprising software        capable of comparing the first and second fluorescent signal.

Furthermore, according to an embodiment of the present invention, thebiomarker is a sepsis biomarker.

Moreover, according to an embodiment of the present invention, thebiomarker is CD64 or CD163.

Additionally, according to an embodiment of the present invention, thesample is a blood sample.

According to another embodiment of the present invention, thefluorescent label of the binding moiety and the fluorescent label of theparticles is the same fluorescent label.

Further, according to an embodiment of the present invention, thebinding moiety is an antibody.

According to an embodiment of the present invention, the software iscapable of recognizing a specific lot of fluorescently-labeledparticles.

Moreover, according to an embodiment of the present invention, theindividual fluorescent signals include at least one first fluorescentsignal and at least one second fluorescent signal.

Additionally, according to an embodiment of the present invention thefluorescently-labeled binding moiety targets a first cell population anda second cell population in the sample.

According to another embodiment of the present invention the detectionof binding of the binding moiety to the second cell population providesan internal positive control for the sample.

Furthermore, according to an embodiment of the present invention, thebinding moiety is anti-CD64 antibody and the first cell populationincludes polymorphonuclear leukocytes.

Yet further, according to an embodiment of the present invention, thesecond cell population includes monocytes.

According to an embodiment of the present invention, the method furthercomprises the step of determining the presence of at least one cellpopulation in the sample that is not bound by the binding moiety, thusproviding an internal negative control for the sample.

There is thus provided according to another embodiment of the presentinvention, a composition for evaluating a biological condition, thecomposition comprising;

-   -   a. a sample comprising at least one of;        -   i. a bodily specimen comprising a target moiety;        -   ii. a positive control moiety; and        -   iii. a negative control moiety;    -   b. an antibody composition comprising at least one of;        -   i. at least one target antibody;        -   ii. at least one positive control identifying antibody; and        -   iii. at least one negative control identifying antibody or            characteristic; and    -   c. at least one reference composition comprising at least one        of;        -   i. a target antibody reference composition; and        -   ii. a reference identifier composition.

According to an embodiment of the present invention, the compositionfurther comprises at least one conditioning moiety comprising;

a) at least one lysis reagent; and

b) at least one diluent.

There is thus provided according to another embodiment of the presentinvention, a method of determining the presence or absence of sepsis ina subject, the method including;

-   -   a) contacting a blood sample from the subject with a        fluorescently-labeled binding moiety specific to a sepsis        marker, wherein the volume of the blood sample is 50 μL or        smaller; and    -   b) detecting the presence, absence or level of the binding        moiety in the sample, thereby determining the presence or        absence of sepsis in the subject.

There is thus provided according to another embodiment of the presentinvention, a method of quantifying a biomarker in a sample, comprising;

-   -   a) contacting the sample with a fluorescently-labeled binding        moiety that specifically binds to the biomarker;    -   b) detecting a first fluorescent signal from at least a portion        of the labeled sample;    -   c) detecting a second fluorescent signal from a population of        fluorescently-labeled particles, wherein the population includes        a known fluorescent intensity over a fixed time; and    -   d) normalizing the first fluorescent signal to the second        fluorescent signal, thereby quantifying the biomarker, wherein        the normalizing includes using a device comprising software        capable of comparing the first and second fluorescent signal.

According to some embodiments, the sample may be liquid, according toother embodiments, the sample may be a colloid or suspension. Accordingto further embodiments, the sample may be a solid, such as in a powderor crystal form.

Typical turnaround times for diagnostic prior art assays are 30-120minutes. Often, the time lost in waiting for laboratory results can leadto a further deterioration in a patient, and sometimes death. In somecases, the physician has to act without having the laboratory results.This can lead to providing the patient with the wrong treatment. Thepresent invention provides rapid assays to save lives and provide fastcorrect treatments to a patient.

There is thus provided according to an embodiment of the presentinvention automated method of determining the presence or absence ofsepsis in a subject, including;

-   -   a) contacting a blood sample from the subject with a        fluorescently-labeled binding moiety specific to a sepsis        marker, wherein the volume of the blood sample is 50 μL or        smaller; and    -   b) detecting the presence, absence or level of the binding        moiety in the sample, thereby determining the presence or        absence of sepsis in the subject within twenty minutes.

Additionally, according to an embodiment of the present invention, thesepsis marker is CD64.

Furthermore, according to an embodiment of the present invention, asecond sepsis marker is CD163.

Moreover, according to an embodiment of the present invention, themethod further includes contacting the blood sample with a secondfluorescently-labeled binding moiety specific for a second sepsismarker.

Further, according to an embodiment of the present invention, the sepsismarker is CD64 and the second sepsis marker is CD163.

Additionally, according to an embodiment of the present invention, thebinding moiety is an antibody.

Moreover, according to an embodiment of the present invention, thedetecting step is performed in a device capable of receiving the sampleand capable of detecting the binding moiety.

Additionally, according to an embodiment of the present invention, themethod further includes the step of calibrating the device by detectinga population of the fluorescently-labeled particles.

According to another embodiment of the present invention, the particlesinclude the same fluorescent label as the fluorescently-labeled bindingmoiety.

Additionally, according to an embodiment of the present invention, themethod further includes a second population of particles that includethe same fluorescent label as the second fluorescently-labeled bindingmoiety.

Moreover, according to an embodiment of the present invention, themethod further includes performing an internal calibration after thedetecting the fluorescently-labeled binding moiety.

Notably, according to an embodiment of the present invention, thecalibration is completed in less than 5 minutes.

According to some embodiments, the particles are microbeads.

Additionally, according to an embodiment of the present invention, themethod is performed in less than 15 minutes.

Furthermore, according to an embodiment of the present invention, themethod, further includes the step of determining the presence of atleast one cell population in the sample that is not bound by the bindingmoiety, thus providing an internal negative control for the sample.

The present invention will be more fully understood from the followingdetailed description of the preferred embodiments thereof, takentogether with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in connection with certain preferredembodiments with reference to the following illustrative figures so thatit may be more fully understood.

With specific reference now to the figures in detail, it is stressedthat the particulars shown are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of the invention. In this regard, noattempt is made to show structural details of the invention in moredetail than is necessary for a fundamental understanding of theinvention, the description taken with the drawings making apparent tothose skilled in the art how the several forms of the invention may beembodied in practice.

In the drawings:

FIG. 1 is a simplified schematic illustration showing an apparatus fordetecting a biological condition, in accordance with an embodiment ofthe present invention;

FIG. 2 is a simplified flow chart of a method for detecting a biologicalcondition, in accordance with an embodiment of the present invention;

FIG. 3 is a simplified schematic illustration showing a methodology fordetecting a biological condition associated with a CD64 cell surfaceantigen, in accordance with an embodiment of the present invention;

FIG. 4 is a simplified flow chart of a method for detecting a biologicalcondition associated with a CD64 cell surface antigen, in accordancewith an embodiment of the present invention;

FIG. 5A is a graphical output of a fluorescent detection assay of anon-activated neutrophil signature associated with the method of FIGS.3-4, in accordance with an embodiment of the present invention;

FIG. 5B is a graphical output of a fluorescent detection assay of anactivated neutrophil signature, associated with the method of FIGS. 3-4,in accordance with an embodiment of the present invention;

FIG. 5C is a graphical output of a fluorescent detection assay of amonocyte signature, associated with the method of FIGS. 3-4, inaccordance with an embodiment of the present invention;

FIG. 5D is a graphical output of a fluorescent detection assay of areference bead signature, associated with the method of FIGS. 3-4, inaccordance with an embodiment of the present invention;

FIG. 6 is a simplified flow chart of a method for differentiatingbetween different particles, in accordance with an embodiment of thepresent invention;

FIG. 7 is a graphical output of fluorescence from reference beads ineight wavebands, in accordance with an embodiment of the presentinvention;

FIG. 8 is a graphical output of data from FIG. 7 after a firstmathematical manipulation, in accordance with an embodiment of thepresent invention;

FIG. 9 is a graphical output of data from FIG. 7 after a secondmathematical manipulation, in accordance with an embodiment of thepresent invention;

FIG. 10 is a graphical output of data from FIG. 7 after a thirdmathematical manipulation, in accordance with an embodiment of thepresent invention; and

FIG. 11 is a graphical output of an event locator, based on data fromFIG. 8-10, in accordance with an embodiment of the present invention.

In all the figures similar reference numerals identify similar parts.

DETAILED DESCRIPTION OF THE INVENTION

In the detailed description, numerous specific details are set forth inorder to provide a thorough understanding of the invention. However, itwill be understood by those skilled in the art that these are specificembodiments and that the present invention may be practiced also indifferent ways that embody the characterizing features of the inventionas described and claimed herein.

International patent application publication no. WO2011/128893 to Kasdanet al., describes a device, system and method for rapid determination ofa medical condition and is incorporated herein by reference.

The microfluidic cartridges of the present invention may be any suitablecartridge as shown in the figures or any of the prior art cartridgesdescribed or cited herein, such as, but not limited to, those describedin U.S. D669191 S1, US20120266986 A1, EP1846159 A2, US2012275972,WO11094577A, US2007292941A and EP1263533 B1.

Reference is now made to FIG. 1, which is a simplified schematicillustration showing an apparatus 100 for detecting a biologicalcondition, in accordance with an embodiment of the present invention.

Apparatus 100 is a kit comprising a cartridge 102 and a number ofchemical/biochemical reactants termed herein, treatment compositions.The treatment compositions are adapted to react, at least in part, withbiological specimen, such as a body specimen, to be introduced to theapparatus. The body specimen may be a bodily fluid such as, but notlimited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid(CSF), serous fluid, peritoneal fluid and synovial fluid. Additionallyor alternatively, the body specimen may be a solid such as a hair, atooth part, a bone part or a piece of cartilage.

Apparatus 100 comprises a specimen receiving element 118, adapted totransfer the specimen to a sample composition chamber 104. The samplecomposition chamber comprises on or more transfer elements 105, adaptedto transfer the specimen from the sample composition chamber to one ormore other locations in the cartridge. In the non-limiting example shownin FIG. 1, transfer element 105 is a conduit in fluid connection with atreatment chamber 112.

Additionally, the cartridge comprises a number of treatment compositionchambers 106, 108, 110, adapted to respectively house a correspondingnumber of treatment compositions 120, 122, 124. These treatmentcompositions may be liquid, solid or combinations thereof. Apparatus 100is typically sold commercially as a kit with the treatment compositionsdisposed therein. In some cases, the apparatus 100 may be adapted for aone-off test and may be disposable. In other cases, the apparatus may bere-used. A re-usable apparatus may be adapted to receive additionalexternal compositions (not shown) or may have a plurality of treatmentcompositions, wherein only a portion is used for each test.

The apparatus may be constructed and configured such that the treatmentcomposition comprises proteins attached to a surface, such as to beads.A plurality of beads or other structural elements with proteins attachedto their surfaces can be made by any one or more of the followingmethodologies:—

-   -   simple attachment such as by adsorption via electrostatic or        hydrophobic interactions with the surface, entrapment in        immobilized polymers, etc.    -   non-covalent or physical attachment;    -   covalent bonding of the protein to the bead surface    -   biological recognition (e. g., biotin/streptavidin).    -   requires two steps: a first layer is formed by silane chemistry        such that the surface presents a reactive group (e. g., epoxy,        amino, thiol, etc.), and a second layer (e. g., the protein to        be immobilized or a linker molecule) is covalently attached via        the immobilized reactive groups.    -   covalent attachment to functionalized polymer coatings on the        interior of the device or linkage to the free end of a        self-assembled monolayer (SAM) on a gold surface.

The reaction type may include any one or more of antigen-antibodybinding, sandwich (such as antibody-antigen-antibody), physicalentrapment, receptor-ligand, enzyme-substrate, protein-protein,aptamers, covalent bonding or biorecognition.

Cartridge 102 further comprises at least one transfer element 107, 109,111 in fluid communication with each respective of treatment compositionchamber, each transfer element also being in fluid communication withtreatment chamber 112. These elements are typically microfluidicchannels and may be designed for mixing, such as being tortuous inshape.

Various methodologies for transferring the contents of the treatmentcomposition chambers and the sample composition chamber via the transferelements to the treatment chamber may be employed, some of which areknown in microfluidics technologies. These include air blowing, suction,vacuuming, mechanical transfer, pumping and the like.

Cartridge 102 further comprises at least one transfer element 113 influid communication with treatment chamber 112 and with an evaluationchamber 114.

Optionally, evaluation chamber 114 is further in fluid communicationwith a transfer element 115, adapted to remove the contents of theevaluation chamber for disposal outside the cartridge. Alternatively,the evaluation chamber may have no external disposal means.

Table 1 shows some representative applications of apparatus 100 andmethods of the present invention.

TABLE 1 Applications of the apparatus and methods of this invention.Typical Prior Art This Relevant Laboratory invention Figures inTurnaround Turnaround Type of this time (TAT)- time Application Testinvention see references (TAT) References Application #1 - Surface FIGS.1-2 and 4 hours 10 U.S. Pat. No. 8,116,984, CD64 Infection & Marker 3-5Dminutes Davis, BH et al., Sepsis (2006) 1 - Fetal Hemoglobin PlasmaFIGS. 1-2 and 4 hours 10 Dziegiel et al. Test Protein 6-8D minutes(2006) 2 - Low Platelet Surface FIGS. 1-2 and 4 hours 10 Segal, H. C.,et al. Count Marker 3-5D minutes (2005): 3 - Resolving BLAST SurfaceFIGS. 1-2 and 4 hours 10 Guerti, K., et al. Flag for hematology Marker3-5D minutes Lab 4 - CD34 Stem Cell Surface FIGS. 1-2 and 4 hours 10Sutherland et al. Enumeration Assay Marker 3-5D minutes (1996) 5 -Platelets Surface FIGS. 1-2 and 4 hours 10 Graff et al. (2002)Activation Assay Marker 3-5D minutes Divers, S. G., et CD62 al. (2003)6 - D-dimer (Bead Plasma FIGS. 1-2 and 4 hours 10 Stein et al. (2004)based protein) Protein 6-8D minutes Rylatt, D. B., et al. (1983): 7 -Chorioamnioitis Surface FIGS. 1-2 and 4 hours 10 Hillier et al. CD64Marker 3-5D minutes (1988) 8 - CD20 Cell Surface FIGS. 1-2 and 4 hours10 Rawstron et al. Quantitation Marker 3-5D minutes (2001) (TherapyMonitoring Cheson et al. (1996) 9 - CD52 Cell Surface FIGS. 1-2 and 4hours 10 Rawstron et al. quantitation (Therapy Marker 3-5D minutes(2001) Monitoring) 10 - Circulating Surface FIGS. 1-2 and 4 hours 10Cristofanilli et al. Tumor Cells Marker 3-5D minutes (2004 11 -Reticulated Surface FIGS. 1-2 and 4 hours 10 Matic et al. Platelet AssayMarker 3-5D minutes (1998) Ault et al (1993) Wang et al. (2002) 12 -Bacteria 4 hours 10 Blajchman et al Detection in platelet minutes (2005)packs McDonald et al. (2005) 13 - Platelet Surface FIGS. 1-2 and 4 hours10 Michelson (1996) Associated Marker 3-5D minutes Antibodies 14 -Residual Surface FIGS. 1-2 and 4 hours 10 Bodensteiner, Leukocyte Countin Marker 3-5D minutes (2003) blood products 15 - CD4 HIV AIDS SurfaceFIGS. 1-2 and 4 hours 10 Rodriguez (2005). Marker 3-5D minutes Dieye etal. (2005) 16 - Leukemia Panels - Surface FIGS. 1-2 and 4 hours 10Drexler et al Very complex Marker 3-5D minutes (1986) 17 - BladderCancer Surface FIGS. 1-2 and 4 hours 10 Ramakumar et al Screening inUrine - Marker 3-5D minutes (1999) Urine sample Lotan et al. (2009) 18 -HLA DR Sepsis Surface FIGS. 1-2 and 4 hours 10 Hershman et al. andMarker 3-5D minutes (2005) Immunosuppression Perry et al (2003) 19 -RECAF Protein Plasma FIGS. 1-2 and 4 hours 10 Moro et al. for Canine andother Protein 6-8D minutes (2005). Cancers 20 - CytoImmun - 4 hours 10Hilfrich et al. Cervical Screening minutes (2008) 21 - ProcalcitoninPlasma FIGS. 1-2 and 4 hours 10 Assicot et al. (Bead Based Protein) +Protein 6-8D minutes (1993) Feasibility Christ-Crain et al. (2004)

Reference is now made to FIG. 2, which is a simplified flow chart 200 ofa method for detecting a biological condition, in accordance with anembodiment of the present invention.

It should be understood that each of the steps of the method may take apredetermined period of time to perform, and in between these stepsthere may be incubation and/or waiting steps, which are not shown forthe sake of simplicity.

In a sample transferring step 202, a sample, such as a bodily specimenis transferred from outside apparatus 100 via receiving element 118 intosample composition chamber 104 and then to the treatment chamber 112.According to some embodiments, the volume of the specimen or sample isless than 200 μL, less than 100 μL, less than 50 μL, less than 25 μL orless than 11 μL.

Thereafter, treatment composition 120 is transferred via transferelement 107 to the treatment chamber in a composition transfer step 204.In some cases, there may be a treatment composition disposed in thetreatment chamber.

Depending on the nature of the treatment composition and sample/specimentype, there may be a requirement to mix or agitate the treatment chambercontents in an optional mixing step 206. This may be performed by usinga small stirbar (not shown) disposed in the chamber. Additionally oralternatively, this may be effected by the fluid dynamics of kit.Additionally or alternatively, stirbars may be disposed in any of theother chambers in the apparatus.

Typically, the total sample volumes are in the range of 10 to 1000 μL,100 to 900 μL, 200 to 800 μL, 300 to 700 μL, 400 to 600 μL, or 420 to500 μL.

According to some embodiments, the volume of the treatment compositionchambers 106, 108, 110 (also called blisters) is from about 1 μL to 1000μL. According to other embodiments, the volume of the specimen is fromabout 10 μL to 200 μL. According to other embodiments, the volume of thespecimen is about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, or 500 μL.

According to some embodiments, the volume of the treatment compositions120, 122, 124 is at most about 500 μL. According to other embodiments,the volume of the specimen is at most about 200 μL. According to otherembodiments, the volume of the specimen at most about 500, 450, 400,350, 300, 250, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30,20, 10, or 1 μL.

According to some embodiments, the volume of a reactant is at leastabout 1 μL. According to other embodiments, the volume of the specimenis from about 10 μL. According to other embodiments, the volume of thespecimen is at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 μL.

The sequence of transfer of the various treatment compositions may beimportant to the reaction sequence and is typically predefined. Steps204-206 may be performed, for example on treatment composition chamber106, thereafter on treatment composition chamber 108 and thereafter ontreatment composition chamber 110. In some cases, some of these stepsmay be performed concurrently.

In a checking step 208, it is ascertained whether all the compositionsrequired for the sample treatment have been transferred to the treatmentchamber. If any compositions remain, then steps 204-206 are performed onthe subsequent treatment composition chamber(s). If no further treatmentcompositions require transfer, then the sample/specimen is transferredfrom chamber 104 into the treatment chamber.

Thereafter, in a second sample transfer step 210, the sample istransferred from the sample composition chamber into the treatmentchamber.

According to some embodiments, step 210 may be performed before steps204-208. If required, an optional mixing step 212 to the contents of thetreatment chamber may be performed.

In a transferring step 214, the contents of the treatment chamber aretransferred to the evaluation chamber.

The evaluation chamber 114 is configured and constructed for one or moreevaluation steps 216. These may include any of the following, orcombinations thereof:

a) transfer of radiation there-through,

b) impinging radiation thereupon;

c) detecting reflected, refracted, and/or transmitted radiation,

d) detecting emitted radiation;

e) capturing one or more images thereof;

f) performing image analysis on the captured images;

g) measuring electrical characteristics of the treated specimen;

h) impinging sonic energy thereon;

i) detecting sonic energy therefrom; and

j) analyzing the outputs of any one or more of the above steps.

According to some embodiments, the cartridge is introduced into a systemas described in International patent application publication no.WO2011/128893 to Kasdan et al., incorporated herein by reference.

The results of the evaluation step are then outputted in a resultsoutputting step 218.

According to some embodiments; the apparatus may have on-board means forshowing a result, such as a colorimetric strip (not shown). Additionallyor alternatively, the results are displayed in a display unit, separateand remote from apparatus 100.

Reference is now made to FIG. 3, which is a simplified schematicillustration showing a methodology 300 for detecting a biologicalcondition associated with a CD64 cell surface antigen, in accordancewith an embodiment of the present invention.

According to some embodiments, the method is carried out in theapparatus shown in FIG. 1 and as described herein. A biologicalspecimen, such as a blood sample, is aspirated via specimen receivingelement 118 to sample composition chamber 104, and then to treatmentchamber 112. The sample is typically of a volume in the range of 10-200μL.

The blood sample is typically whole blood recently removed from apatient. The whole blood comprises mainly red blood cells (also calledRBCs or erythrocytes), platelets and white blood cells (also calledleukocytes), including lymphocytes and neutrophils. Increased number ofneutrophils, especially activated neutrophils are normally found in theblood stream during the beginning (acute) phase of inflammation,particularly as a result of bacterial infection, environmental exposureand some cancers.

A cocktail 304 comprising antibodies to CD64 and antibodies to CD163 isintroduced to the treatment chamber (see Davis et al. (2006)). Eachantibody type is typically tagged by a specific fluorescent tag.

The contents of the chamber are incubated and/or mixed as is required tobind the activated blood neutrophils with the CD64 tagged antibody (alsocalled a marker) to form activated neutrophils with CD64 marker 310,and/or monocyte with a CD64 tagged antibody and a CD163 tagged antibody312. Lymphocytes with no markers 314 are present in the contents, aswell as unaffected RBCs 316.

Thereafter, a lysis reagent or diluent 306 is introduced into treatmentchamber 112. In the case of a lysis reagent, it is adapted to lyse redblood cells to form lysed red blood cells 324. Additionally,reference/calibration beads 308 are added to the treatment chamber.These are used to calibrate the outputs, as is explained with referenceto FIGS. 5A-5D hereinbelow.

CD64 (Cluster of Differentiation 64) is a type of integral membraneglycoprotein known as an Fc receptor that binds monomeric IgG-typeantibodies with high affinity. Neutrophil CD64 expression quantificationprovides improved diagnostic detection of infection/sepsis compared withthe standard diagnostic tests used in current medical practice.

CD163 (Cluster of Differentiation 163) is a human protein encoded by theCD163 gene. It has also been shown to mark cells of monocyte/macrophagelineage.

Reference is now made to FIG. 4, which is a simplified flow chart 400 ofa method for detecting a biological condition associated with a CD64cell surface antigen, in accordance with an embodiment of the presentinvention.

According to some embodiments, the method is carried out in theapparatus shown in FIG. 1 and as described herein. In a firsttransferring step 402, a biological specimen, such as a blood sample isaspirated via specimen receiving element 118 to sample compositionchamber 104. The sample is typically of a volume in the range of 10-200μL.

Typically, the total sample volumes are in the range of 10 to 1000 μL,100 to 900 μL, 200 to 800 μL, 300 to 700 μL, 400 to 600 μL, or 420 to500 μL.

According to some embodiments, the volume of the treatment compositionchambers 106, 108, 110 (also called blisters) is from about 1 μL to 1000μL. According to other embodiments, the volume of the specimen is fromabout 10 μL to 200 μL. According to other embodiments, the volume of thespecimen is about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, or 500 μL.

According to some embodiments, the volume of the treatment compositions120, 122, 124 is at most about 500 μL. According to other embodiments,the volume of the specimen is at most about 200 μL. According to otherembodiments, the volume of the specimen at most about 500, 450, 400,350, 300, 250, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30,20, 10, or 1 μL.

According to some embodiments, the volume of a reactant is at leastabout 1 μL. According to other embodiments, the volume of the specimenis from about 10 μL. According to other embodiments, the volume of thespecimen is at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 μL.

In an addition step 404, a cocktail of tagged antibodies to CD64 and toCD163 is added to the treatment chamber 112 and is incubated with theblood sample. In the incubation phase of this step, the antibodies bindactivated neutrophils with CD64 marker 310, and/or monocytes activatedwith a CD64 tagged antibody and a CD163 tagged antibody 312.

In a lysis reagent addition step 406, the lysis reagent is added to thetreatment chamber and thereby lyses at least some of the RBCs in thechamber.

At any suitable time, typically following lysis step 406, referencebeads are added to the contents of the treatment chamber in a referencebead adding step 408.

After a predefined period of time, an analysis step 410 is performed toanalyze the fluorescent emission signatures from the contents. This isdescribed in further detail with reference to FIGS. 5A-5D. According tosome examples, the evaluation chamber 114 is constructed and configuredto allow cells to pass through a reading zone 130 such that each cellpassing therethrough is analyzed individually. The assay sensitivity isaround 86% and its specificity is around 87% (Hoffmann, 2011).

The time required to complete an assay using apparatus 100 of thepresent invention varies depending on a number of factors, withnon-limiting examples that include described herein. In someembodiments, the time required to complete an assay is from about 0.5 to100 minutes. In other embodiments, the time required to complete anassay is from about 1 to 20 minutes. In still other embodiments, thetime required to complete an assay is from about 1 to 10 minutes. Insome examples, the time required to complete an assay is from about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80, or 100minutes.

Reference is now made to FIG. 5A, which is a graphical output of afluorescent detection assay of a non-activated neutrophil signature 500associated with the method of FIGS. 3-4, in accordance with anembodiment of the present invention. The non-activated taggedneutrophils each emit a signal 502 at wavelength W1 of an intensity I1.The wavelengths shown in FIGS. 5A-5D represent a peak wavelength ofwaveband outputs detected, as are shown in FIGS. 7-11.

FIG. 5B shows a graphical output of a fluorescent detection assay of anactivated neutrophil signature 510, associated with the method of FIGS.3-4, in accordance with an embodiment of the present invention. Eachactivated tagged neutrophil emits an activated neutrophil signature 512at wavelength W1 of an intensity I2. Typically I2 is greater than I1. Insome cases the difference in signatures 512 and 510 may be detected byan image analysis, a fluorescent emission radiation count or by otherqualitative or quantitative methods known in the art. The currentexample is not meant to be limiting.

Turning to FIG. 5C, there can be seen a graphical output of afluorescent detection assay of a monocyte signature 520, associated withthe method of FIGS. 3-4, in accordance with an embodiment of the presentinvention. The monocyte signature comprises a first signal 522 at afirst wavelength W1 of an intensity 13 and a second signal 524 at asecond wavelength W2 of an intensity 14.

FIG. 5D shows a graphical output of a fluorescent detection assay of areference bead signature 530, associated with the method of FIGS. 3-4,in accordance with an embodiment of the present invention. The referencebead signature comprises a first signal 532 at a first wavelength W1 ofan intensity I1 (similar or equal to non-activated tagged neutrophils'signal 502) and a second signal 534 at a second wavelength W3 of anintensity IS.

This methodology enables the identification and quantification ofactivated neutrophils by intensity of signature 512 of the CD64 tag.Monocytes are identified by the double signal signature 522, 524, actingas a positive control. Reference beads are identified by the uniquesignal 534 at wavelength W3. The intensity of signal 532 at wavelengthW1 provides a reference level of CD64 for the comparison of intensity of512 of the neutrophils.

Lymphocytes with no markers 330 (FIG. 3) act as a negative control andshould provide no fluor signature, but may be detected by theirscattering or other characteristics. Further details of some embodimentof this assay procedure are described in U.S. Pat. No. 8,116,984 and inDavis, B H et al., (2006).

Reference is now made to FIG. 6, which is a simplified flow chart of amethod 600 for differentiating between different particles, inaccordance with an embodiment of the present invention.

The input to the processing is a time series from each of the channelsin the eight channel photomultiplier array 601. In addition, data frommultiple scatter channels 609 is introduced. Each fluorescent timeseries and scatter time series may be processed individually employingrespective spectral crosscorrelation algorithm 606 and scatter algorithm607 to smooth it and minimize noise. Two possible processing methods areboxcar averaging algorithm 602 and matched filtering algorithm 604. Inaddition, groups of individual channels may be correlated to yield amultiple spectral crosscorrelations 606. One or more of these derivedtime series may be used to determine event locations.

Once an event is located in the eight channel time series thecomposition of that event in terms of known fluorophore signatures isdetermined using a minimum mean square error fit 610. The event is nowdescribed in terms of its composition of known fluors. Each event thusdescribed is stored in an event store, i.e. memory, together with thedata from the eight time series for that event and its description 612.Based on the fluor composition for each event in the data store, it ispossible to determine the type of particle. For example, a neutrophil616 is characterized by the single fluor attached to the CD64 antibodyshown in FIG. 5 as W1. Thus events that are preponderantly characterizedby the single fluor attached to the CD64 antibody are identified asneutrophils.

Similarly, monocytes 618 are characterized by fluors W1 and W2 so thatan event with both of these fluor signatures is identified as amonocyte. Similarly, a bead 620 is characterized by an event that hasfluors W1 and W3. Lymphocytes 622 do not express significantfluorescence but are identified by their scatter as events. Events thatdo not match any of the known combinations of the fluorophores areidentified as rejects 626.

Given the population of identified events, the median intensity of theneutrophil population and the median intensity of the bead populationare determined. The ratio of the neutrophil median to the bead median isthe desired Leuko64 index. The positive control value is determined asthe median intensity of the CD64 fluorophore bound to monocytes dividedby the median intensity of the same fluorophore on the bead population.The negative control value is determined by the median intensity of theCD64 fluorophore bound to lymphocytes. These are the key steps inperforming the Leuko64 assay.

FIG. 7 is a graphical output 700 of fluorescence from reference beads ineight wavebands, in accordance with an embodiment of the presentinvention. This figure shows the smoothed signals from the eight channelPMT array for two reference beads. The amplitude for each waveband isshown on the same graph. The corresponding wavelength range is shown foreach plot 702, 706, 708, 710, 712, 714, 716, 718 in the legend box. Thetwo fluorophores signatures present in this plot are 702, 706 and 708for FITC, which is the fluorophore attached to the CD64 antibody and710, 712 for Starfire Red, which is the fluorophore identifying thereference beads.

Reference is now made to FIG. 8, which is a graphical output 800 of datafrom FIG. 7 after a first mathematical manipulation, in accordance withan embodiment of the present invention. FIG. 8 shows the crosscorrelation of wave bands one two and three corresponding to wavelength500 to 525, 525 to 550, and 552 to 575 nm. This cross correlation iscomputed by multiplying the boxcar smoothed time series corresponding tothese wavelengths. This signal will have a high-value when an eventcontaining the FITC fluorophore is present.

FIG. 9 is a graphical output 900 of data from FIG. 7 after a secondmathematical manipulation, in accordance with an embodiment of thepresent invention. FIG. 9 shows the cross correlation of wave bands 3, 4and 5 corresponding to wavelengths 550 to 575, 575 to 600, and 600 to625 nm. This signal will have a high-value when an event containing thePE fluorophore is present.

FIG. 10 is a graphical output 1000 of data from FIG. 7 after a thirdmathematical manipulation, in accordance with an embodiment of thepresent invention. FIG. 10 shows the cross correlation of wave bands 7and 8 corresponding to wavelengths 650 to 675, and 675 to 700 nm. Thissignal will have a high-value when an event containing the Starfire Redfluorophore is present.

FIG. 11 is a graphical output 1100 of an event locator, based on datafrom FIG. 8-10, in accordance with an embodiment of the presentinvention. FIG. 11 shows the event locations determined from the crosscorrelations computed in FIGS. 8, 9 and 10. The solid fill area 1102corresponds to the region where any of the cross correlations 802, 902and 1002 exceeded a predefined threshold. Similarly, the solid fill area1104 corresponds to the region where any of the cross correlations 804,904 and 1004 exceeded a predefined threshold. This then completes theevent location process.

Example

Application No. 1—CD64 Infection & Sepsis

A cartridge 102 (FIG. 1) is prepared for receiving a blood sample. Thecartridge comprises a number of treatment composition chambers 106, 108,110, adapted to respectively house a corresponding number of treatmentcompositions 120, 122, 124. These compositions are described in furtherdetail in U.S. Pat. No. 8,116,984 and in Davis, B H et al., (2006)),incorporated herein by reference. In brief, Reagent A comprises amixture of murine monoclonal antibodies (contains buffered saline),Reagent B—10× Concentrated Trillium Lyse solution (contains ammoniumchloride), Reagent C—suspension of 5.2 μm polystyrene beads labeled withStarfire Red and fluorescein isothiocyanate (FITC), (contains <0.1%sodium azide and 0.01% Tween 20).

In a sample transferring step 202 (FIG. 2), a 10 uL blood sample, istransferred from outside apparatus 100 via receiving element 118 intosample composition chamber 104 and then on to treatment chamber 112 in atransferring step 214.

An antibody composition (Reagent A) 120 comprising CD64 antibodies istransferred via transfer element 107 to the treatment chamber 112 in acomposition transfer step 204.

These two steps combined with mixing step 206 take around four minutesusing cartridge 102 of the present invention.

A lysis buffer (Reagent B) 122 is also added and mixed with theresultant mixed composition. This step and mixing all the compositionstakes around three minutes using cartridge 102 of the present invention.Reference beads (Reagent C) 308 are added to the treatment chamber.

The evaluation chamber 114 is configured and constructed for one or moreevaluation steps 216.

According to some embodiments, the cartridge is introduced into a systemas described in International patent application publication no.WO2011/128893 to Kasdan et al., incorporated herein by reference. Thissystem has software associated therewith for computing the CD64 andCD163 indices on leukocytes.

The results of the evaluation step are then outputted in a resultsoutputting step 218. According to this example, the time taken from theintroduction of the small blood sample to obtaining an indication ofsepsis is less than 15 minutes, typically around 10 minutes.

From a user point of view, the following steps are performed:

-   -   1) The user adds drop of blood to the cartridge 102 and seals        it. (10 μL are metered out by microfluidics).    -   2) Blister A (106) is pressed, releasing 100 μL of Reagent A.        Mixing in the cartridge is controlled by the cartridge handling        unit (CHU), followed by a 4-minutes incubation.    -   3) Blister B (108) is pressed, releasing ˜250 μL of Reagent B.        Mixing in the cartridge is controlled by the CHU, followed by a        3-5-minutes incubation.    -   4) Magnetic stirbar is activated, stirring the bead suspension        (Reagent C).    -   5) Blister C (110) is pressed, releasing 100 μL of Reagent C.        Mixing in the cartridge is controlled by the CHU. According to        one example, Reagent A is a mixture of murine monoclonal        antibodies—diluted 1:5 in buffered saline (PBS+0.5% BSA);        Reagent B is a Trillium Lyse solution (at working        concentration); Reagent C is a suspension of 5.2 μm polystyrene        beads labeled with Starfire Red and FITC, diluted 1:100 in        PBS+0.01% Tween 20.    -   6) The sample is read by the optoelectronics core, and collected        to the reading below.    -   7) Data is analyzed automatically and result is presented.    -   8) The cartridge is disposed as biohazard.

TABLE 2 Comparison of Prior art methodology with the methodology of thepresent invention for detecting sepsis using CD64 and CD163 antibodies.LeukoDx device-present invention Step Description Volume (uL) Duration(min) comments 1 Mixing blood and Blood - 10 4 antibodies Abs - 50 2Adding RBC 250 3 Might lysis buffer require heating the buffer to 37 C.3 Incubating, Vortexing 3 4 Adding normalization 2 Less than 1 beads 5Reading Less than 1 Total 312 10 

In the case of sepsis, by “normalization” is meant taking the ratio ofthe median of the target population fluorescence emission to the medianof the reference bead population fluorescence emission.

According to some embodiments, the readout may comprise anoptoelectronics core, which enables identification and detection offluorescent signals.

The CCD in the core, used for focusing, can also be used to readchemiluminescent signals. The readout to user may also indicate wherethe result falls relative to reference ranges.

The contents of these publications are incorporated by reference hereinwhere appropriate for teachings of additional or alternative details,features and/or technical background.

It is to be understood that the invention is not limited in itsapplication to the details set forth in the description contained hereinor illustrated in the drawings. The invention is capable of otherembodiments and of being practiced and carried out in various ways.Those skilled in the art will readily appreciate that variousmodifications and changes can be applied to the embodiments of theinvention as hereinbefore described without departing from its scope,defined in and by the appended claims.

REFERENCES

-   Assicot, Marcel, et al. “High serum procalcitonin concentrations in    patients with sepsis and infection.” The Lancet 341.8844 (1993):    515-518.-   Aulesa, C., et al. “Validation of the Coulter LH 750 in a hospital    reference laboratory.” Laboratory Hematology 9.1 (2003): 15-28.-   Ault, Kenneth A. “Flow cytometric measurement of platelet function    and reticulated platelets.” Annals of the New York Academy of    Sciences 677.1 (1993): 293-308.-   Blajchman, Morris A., et al. “Bacterial detection of platelets:    current problems and possible resolutions.” Transfusion medicine    reviews 19.4 (2005): 259-272.-   Bodensteiner, David C. “A flow cytometric technique to accurately    measure post-filtration white blood cell counts.” Transfusion 29.7    (1989): 651-653.-   Cheson, Bruce D., et al. “National Cancer Institute-sponsored    Working Group guidelines for chronic lymphocytic leukemia: revised    guidelines for diagnosis and treatment.” Blood 87.12 (1996):    4990-4997.-   Christ-Crain, Mirjam, et al. “Effect of procalcitonin-guided    treatment on antibiotic use and outcome in lower respiratory tract    infections: cluster-randomised, single-blinded intervention trial.”    Lancet 363.9409 (2004): 600-607.-   Cristofanilli, Massimo, et al. “Circulating tumor cells, disease    progression, and survival in metastatic breast cancer.” New England    Journal of Medicine 351.8 (2004): 781-791.-   Davis, Bruce H., et al. “Neutrophil CD64 is an improved indicator of    infection or sepsis in emergency department patients.” Archives of    pathology & laboratory medicine 130.5 (2006): 654-661.-   Dieye, Tandakha Ndiaye, et al. “Absolute CD4 T-cell counting in    resource-poor settings: direct volumetric measurements versus    bead-based clinical flow cytometry instruments.” JAIDS Journal of    Acquired Immune Deficiency Syndromes 39.1 (2005): 32¬37.-   Divers, S. G., et al. “Quantitation of CD62, soluble CD62, and    lysosome-associated membrane proteins 1 and 2 for evaluation of the    quality of stored platelet concentrates.” Transfusion 35.4 (2003):    292-297.-   Drexler, Hans G., et al. “Diagnostic value of immunological leukemia    phenotyping.” Acta haematologica 76.1 (1986): 1-8.-   Dziegiel, Morten Hanefeld, Leif Kofoed Nielsen, and Adela Berkowicz.    “Detecting fetomaternal hemorrhage by flow cytometry.” Current    opinion in hematology 13.6 (2006): 490.-   Fischer, Johannes C., et al. “Reducing costs in flow cytometric    counting of residual white blood cells in blood products:    utilization of a single platform bead free flow rate calibration    method.” Transfusion 51.7 (2011): 1431-1438.-   Graff, Jochen, et al. “Close relationship between the platelet    activation marker CD62 and the granular release of platelet-derived    growth factor.” Journal of Pharmacology and Experimental    Therapeutics 300.3 (2002): 952-957.-   Guerti, K., et al. “Performance evaluation of the PENTRA 60C+    automated hematology analyzer and comparison with the ADVIA 2120.”    International journal of laboratory hematology 31.2 (2009): 132-141.-   Hawkins, Robert C. “Laboratory turnaround time.” The Clinical    Biochemist Reviews 28.4 (2007): 179.-   Hershman, M. J., et al. “Monocyte HLA-DR antigen expression    characterizes clinical outcome in the trauma patient.” British    Journal of Surgery 77.2 (2005): 204-207.-   Hilfrich, Ralf, and Jalil Hariri. “Prognostic relevance of human    papillomavirus L1 capsid protein detection within mild and moderate    dysplastic lesions of the cervix uteri in combination with p16    biomarker.” Analytical and Quantitative Cytology and Histology 30.2    (2008): 78-82.-   Hillier, Sharon L., et al. “A case-control study of chorioamnionic    infection and histologic chorioamnionitis in prematurity.” New    England Journal of Medicine 319.15 (1988): 972-978.-   Hoffmann, Johannes J M L. “Neutrophil CD64 as a sepsis biomarker.”    Biochemia Medica 21.3 (2011): 282-290.-   Kibe, Savitri, Kate Adams, and Gavin Barlow. “Diagnostic and    prognostic biomarkers of sepsis in critical care.” Journal of    Antimicrobial Chemotherapy 66. suppl 2 (2011): ii33-ii40.-   LaRosa, Steven P., and Steven M. Opal. “Biomarkers: the future.”    Critical care clinics 27.2 (2011): 407.-   Liu, N. I. N. G., A. H. Wu, and Shan S. Wong. “Improved quantitative    Apt test for detecting fetal hemoglobin in bloody stools of    newborns.” Clinical chemistry 39.11 (1993): 2326-2329.-   Lotan, Yair, et al. “Bladder cancer screening in a high risk    asymptomatic population using a point of care urine based protein    tumor marker.” The Journal of urology 182.1 (2009): 52-58.-   Masse, M., et al. “Validation of a simple method to count very low    white cell concentrations in filtered red cells or platelets.”    Transfusion 32.6 (2003): 565-571.-   Matic, Goran B., et al. “Whole blood analysis of reticulated    platelets: improvements of detection and assay stability.” Cytometry    34.5 (1998): 229-234.-   McDonald, C. P., et al. “Use of a solid-phase fluorescent cytometric    technique for the detection of bacteria in platelet concentrates.”    Transfusion Medicine 15.3 (2005): 175-183.-   Michelson, Alan D. “Flow cytometry: a clinical test of platelet    function.” Open Access Articles (1996): 290.-   Miller, E. M.; Freire, S. L. S.; Wheeler, A. R. “Proteomics in    Microfluidic Devices” In Encyclopedia of Micro- and Nanofluidics;    Li, D. Q., Ed.; Springer: Heidelberg, Germany, 2008; Vol. 3, pp    1749-1758.”-   Moro, Ricardo, et al. “A new broad-spectrum cancer marker.” Vitro    Diagnostic Technology (2005).-   Perry, Sara E., et al. “Is low monocyte HLA-DR expression helpful to    predict outcome in severe sepsis?.” Intensive care medicine 29.8    (2003): 1245-1252.-   Ramakumar, Sanjay, et al. “Comparison of screening methods in the    detection of bladder cancer.” The Journal of urology 161.2 (1999):    388-394.-   Rawstron, Andy C., et al. “Quantitation of minimal disease levels in    chronic lymphocytic leukemia using a sensitive flow cytometric assay    improves the prediction of outcome and can be used to optimize    therapy.” Blood 98.1 (2001): 29-35.-   Rodriguez, William R., et al. “A microchip CD4 counting method for    HIV monitoring in resource-poor settings.” PLoS medicine 2.7 (2005):    e182.-   Rylatt, D. B., et al. “An immunoassay for human D dimer using    monoclonal antibodies.” Thrombosis research 31.6 (1983): 767-778.-   Sacks, David B., et al. “Guidelines and recommendations for    laboratory analysis in the diagnosis and management of diabetes    mellitus.” Clinical Chemistry 48.3 (2002): 436-472.-   Segal, H. C., et al. “Accuracy of platelet counting haematology    analysers in severe thrombocytopenia and potential impact on    platelet transfusion.” British Journal of Haematology 128.4 (2005):    520-525.-   Stein, Paul D., et al. “D-dimer for the exclusion of acute venous    thrombosis and pulmonary embolism: a systematic review.” Annals of    internal medicine 140.8 (2004): 589.-   Sutherland, D. Robert, et al. “The ISHAGE guidelines for CD34+ cell    determination by flow cytometry.” Journal of hematotherapy 5.3    (1996): 213-226.-   Wang, Chao, et al. “Reticulated platelets predict platelet count    recovery following chemotherapy.” Transfusion 42.3 (2002): 368-374.

What is claimed is:
 1. A system for analyzing a sample, the systemcomprising: a test cartridge comprising: a) a sample composition chamberadapted for receiving the sample; b) a first, second, and third blistercomprising a cell lysis reagent and a diluent; c) a treatmentcompartment adapted for fluid mixing, wherein the treatment compartmentis coupled to the sample composition chamber, the first blister, thesecond blister, and the third blister via one or more transfer elements,wherein the one or more transfer elements are microfluidic channels thatallow fluid communication between the treatment compartment and (1) thesample composition chamber, (2) the first blister, (3) the secondblister, and (4) the third blister; and d) an evaluation chamber fluidlyconnected to the treatment chamber and comprising a reading zone,wherein the reading zone allows the sample to be analyzed as it passestherethrough; and the sample, wherein the sample comprises beadscomprising a fluorescent tag, fluorescently tagged antibodies, andcells.
 2. The system of claim 1, further comprising a detector operablycoupled to the reading zone.
 3. The system of claim 2, wherein thedetector is a photomultiplier array.
 4. The system of claim 3, whereinthe detector is an eight channel photomultiplier array.
 5. The system ofclaim 1, wherein the reading zone is configured to allow each cellpassing therethrough to be analyzed individually.
 6. The system of claim1, wherein the test cartridge comprises a pump or an air blowing elementcoupled to the treatment compartment.
 7. The system of claim 1, whereinthe pump is a bellow pump.
 8. The system of claim 1, wherein a volume ofeach of the first, second, and third blister is from about 1 microliterto 1000 microliters
 9. The system of claim 1, further comprising acartridge handling unit configured to receive the cartridge.
 10. Thesystem of claim 9, wherein the cartridge handling unit is programmed tocontrol actuation of the first, second, or third blister.
 11. The systemof claim 9, wherein the cartridge handling unit is programmed to controlmixing of the sample on the cartridge.
 12. The system of claim 9,wherein the cartridge handling unit is programmed to complete an assayrunning on the cartridge in a period of about 30 minutes.
 13. The systemof claim 9, wherein the cartridge handling unit is operably coupled to adisplay.
 14. The system of claim 13, wherein the display is configuredto output a graphical output of a fluorescent detection assay relatingto the sample.
 15. The system of claim 1, wherein a volume of the sampleis about 50 microliters or smaller.
 16. The system of claim 1, whereinthe antibodies are murine monoclonal antibodies.
 17. The system of claim1, wherein the cell lysis reagent comprises ammonium chloride.
 18. Thesystem of claim 1, wherein the treatment compartment comprises atortuous shaped channel.
 19. The system of claim 1, wherein the cellscomprise erythrocytes or leukocytes.
 20. The system of claim 1, whereinthe diluent comprises saline.